Amphotericin B is a macrocyclic, polyene antibiotic produced by Streptomycetes nodosus. It is effective against a broad variety of fungi, yeasts and some protozoans.
Amphotericin B for intravenous administration was originally available in a conventional colloidal form. Even today, about 35 years after its development, it is widely used as an important antifungal agent because of its reliable therapeutic efficacy. The tolerability of the drug is low in view of number of adverse effects reported when used clinically. Nephrotoxicity occurs in almost all patients receiving conventional Amphotericin B intravenously. The other adverse effects include hypertension, hypotension, cardiac arrhythmia including ventricular fibrillation, cardiac arrest, liver disorders. Both tubular and glomerular damage occurs and there is a risk of permanent impairment of renal function. Solutions of Amphotericin B irritate the venous endothelium and may cause pain and thrombophlebitis at the injection site because of the synthetic surfactant sodium deoxycholate used in the preparation to solubilise Amphotericin B.
To reduce the toxic effects, Amphotericin B has been formulated in different drug delivery systems such as lipid complex, liposomes and emulsion. These compositions have greater efficacy, yet have a lower toxicity in comparison with the drug when used in the free form. Both the lipid complex and liposomal formulations of Amphotericin B are now available in the market and are approved in various countries worldwide.
The main disadvantage of lipid formulations based on lipid complex and liposome, is the high cost of therapy. Amphotericin B is a lipophilic drug that binds to sterols and intercalates into lipid bilayers and hence Amphotericin B is particularly suitable for use with the lipid-based delivery systems.
An attempt in this laboratory was made to formulate Amphotericin B in the form of a lipid based emulsion which have the benefits of low toxicity at lower cost of therapy.
Volker Heinemann et. al. have postulated [Antimicrobial agents and chemotherapy 1997, 41(4); 728–732] that the lipid emulsions decrease the amount of oligomeric Amphotericin B and thereby reduce the interaction of Amphotericin B with cholesterol of human cell membranes. The remaining monomeric Amphotericin B however retains its potential to bind to the ergosterol of fangal cell membranes.
Kirsh R. Goldstein R, Tarloff J, and et. al have reported (J. Infect. Dis. 1988, 158; 1065–1070) that the lipid emulsion composition of Amphotericin B prepared by mixing with fat emulsion have low toxicity without loss of antifungal activity. However, the physical stability of such lipid emulsion carrying Amphotericin B has been found to be poor.
Moreau P, et. al have reported (J. Antimicro. Chemother. 1992, 30; 535–541) that patients treated with fat emulsion mixed with Amphotericin B injection appeared to have a significant reduction in infusion related toxicity and renal dysfunction.
The use of Amphotericin B mixed with fat emulsion of parenteral nutrition are increasing both in Europe and United States.
The prior art processes of making Amphotericin B emulsion are discussed below:    U.S. Pat. No. 5,364,632 (1994)/Japanese Patent JP 2290809 (1990)
The process or preparation of an emulsion in accordance with this invention is described in a typical example as follows:
Amphotericin B was dissolved in methanol (0.8 mg/ml) by bath sonication (15 minutes). Phospholipids E-80 (containing mainly 80% phosphatidyl choline and 8% phosphatidyl ehanolamine) were dissolved in chloroform. Both solutions were mixed and filtered through a combined filtering system comprising a fibre glass prefilter and 0.45μ regenerated cellulose membrane filter (RC 5) (GF92) for removing pyrogens and aggregates. The resulting clear lipid solution was deposited as thin film on the walls of a round bottom flask by rotary evaporation under reduced pressure at 40° C. The aqueous phase comprising the poloxamer, sodium deoxycholate and glycerin was filtered through a 0.22 μ Millipore filter, poured into the flask and the dispersion was sonicated until a homogenous liposomal mixture was achieved.
MCT (Medium chain triglyceride) oil, filtered through 0.22 μ Millipore filter, and containing α-tocopherol was heated to 70° C. and then admixed into the liposomal mixture heated to 45° C. and dispersed therein by a magnetic stirrer.
Emulsification was carried out while maintaining the same temperature using a high shear mixer, Polytron. The resulting coarse emulsion was cooled rapidly. A fine monodispersed emulsion was achieved using a two-stage homogeniser.
Finally, the pH of the emulsion was adjusted and was filtered through a 0.45 μ Mllipore filter to discard coarse droplets and debris generated during the emulsification and homogenisation process.
All processing operations were carried out under aseptic conditions.
The relative amounts of the various ingredients in the final emulsions in the Example and the range given in the description are as follows:
Amphotericin B 0.075% (0.015–0.15%), MCT oil 20% (3–50%), phospholipid E80 0.5% (0.5–20%), poloxamer 2% (0.3–10%), sodium deoxycholate 1% (0.5–5%), glycerin 2.25%, α-tocopherol 0.02% and double distilled water 200%.
The drawbacks of U.S. Pat. No. 5,364,632 (1994)/Japanese Patent JP 2290809 (1990) process are:
    i. As the solubility of Amphotericin B in methanol is low, a large amount of methanol is required to dissolve the required quantity of Amphotericin B. This restricts the level of the drug in the final composition.    ii. In this process it is necessary to first form a thin film of the drug, Amphotericin B and phospholipids, and then to hydrate that film using the aqueous phase. The aqueous phase contains non-ionic emulsifying agent poloxamer, surfactant sodium deoxycholate and glycerin.    iii. The oil phase used is MCT oil with added α-tocopherol. The emulsion is prepared by adding the oily phase maintained at 70° C. to aqueous phase maintained at 45° C. This does not ensure that the Amphotericin B is retained in the oil phase. This process does not exploit the full potential of reducing the toxicity of the Amphotericin B if it were to be placed in the oily phase.    iv. The product of the process of U.S. Pat. No. 5,364,632 (1994) is made under aseptic conditions to render it sterile. The preferred process of sterilisation specified in pharmacopoeias is autoclaving of the product in the final container. Further as Amphotericin B is commonly administered by intravenous route, terminal sterilisation is the only preferred alternative which offers higher confidence of sterility compliance.    v. The emulsion product eventhough is stable to mechanical stress, has not been studied for toxicity. The toxicity of the product hence is not known. However in-vivo comparative studies have been done in Balb/c mice in comparison with Fungizone, which is a commercial Amphotericin B formulation containing Sodium deoxycholate. This study indicated that the product is less toxic than Fungizone.    vi. The use of MCT oil and the poloxamer increases the plasma concentration of the drug by reducing the uptake of the drug by reticulo endothelial system (RES). In case of fungal infections, Amphotericin B is required to be distributed in the reticulo-endothelial system, which is the site of infection.Japanese Patent 11-60491 (1989)
In this Japanese Patent a medicinal formulation containing Amphotericin B in the emulsion form has been described. The emulsion contains    i) Amphotericin B (1 to 10 mg/ml of final emulsion).    ii) the oily phase—The oily phase consists of plant oils, fish oil or triglycerides, (1–50%, preferably 5–30%). The preferred oil used is soybean oil or sesame oil.    iii) emulsiflers—The emulsifier used are phospholipids. Additionally non-toxic emulsifiers are also employed. Phospholipids used are such as egg yolk phospholipids, soybean phospholipids, or hydrogenated product obtained from these materials. Phosphatidylcholine, Phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, phosphatidic acid, phosphatidyl glycerol have also be used. The quantity recommended is 1–50% by weight of the oil component, preferably 10–30% by weight of the oil component or 1–10% w/v, preferably 4–6% w/v of the emulsion.            Non-ionic emulsifiers such as polyalkylene glycol (mol. wt. 1000–10000, preferably 4000–6000); or polyoxyethylene or polyoxy propylene polymer) (mol. wt. 1000–20000, preferably 2000–10000), hydrogenated castor oil polyoxy alkylene derivatives such as hydrogenated castor oil polyoxyethylene-20-ether, -40-ether, -100-ether less than 5% w/v preferably less than 1% w/v are employed. A combination of the two non-ionic emulsifiers can also be used.            iv) fatty acids and their salts (Pharmaceutically acceptable)—upto 1% preferably 0.5% w/v.    v) stabilisation agents less than 5% w/v, preferably less than 1% w/v which include            a) high molecular weight polymer substances such as albumin of human origin; vinyl copolymer e.g. polyvinyl pyrolidone, polyvinyl alcohol; aliphatic amines.        b) gelatin, hydroxy ethyl starch; Cholesterol varieties,            vi) isotonic agents—less than 5% w/v preferably less than 1% w/v.    vii) glycerin or its monoesters like mono olein, mono palmitin;    viii) saccharides such as mono and disaccharides, sorbitol, xylitol less than 5% w/v preferably less than 1% w/v.    ix) antioxidants such as tocopherol less than 5% w/v preferably less than 1% w/v;    x) pH controlling agents such as acids, alkalies and buffers.
The process of manufacturing followed is said as reported in the past. This process involves first forming water-in-oil emulsion (w/o) and then converting it into oil-in-water emulsion (o/w) by dilution with water. In this process, soybean oil, phospholipid, Amphotericin B and some water as well as other additives (whenever used) are all mixed together and heated if required. The mixture is then homogenised in high-pressure homogeniser. More water is added in the required quantity to convert w/o emulsion into an o/w emulsion, which is homogenised again.
In a typical example, 200 g of soybean oil, 50 g phospholipid and 2.5 g Amphotericin B and 750 ml water are processed as above.
In another example glycerin 2.2% w/v of the composition has been incorporated in the above composition.
The mean emulsion droplet size is from 0.1–0.2μ. The emulsion and its droplet size is stable up to 10 days under refrigeration conditions.
Drawbacks of this Japanese Patent 11-60491 (1989) process are:
It is known that Amphotericin B in emulsion formulation is less toxic than the conventional Amphotericin B formulation. However, the formulations of this Japanese Patent 11-60491 (1989) does not exploit the full potential of reducing toxicity available to this emulsion concept, because of the process of making the emulsion followed in this patent.    i. Because of the mean particle size of the emulsion obtained in the Japanese patent JP 11-60491 (1989) is from 0.1 to 0.2μ, it is unable to exploit the well-known benefit of the preferential uptake of particles larger in size by reticuloendothelial system. Preferential uptake of Amphotericin B by reticuloendothelial system is required, as this is the site for most of the fungal infections.    ii. The stability of the emulsion is studied up to 10 days in refrigerator.    iii. A large number of additives which include emulsification supporting agents such as aliphatic amines, high molecular wt. polymers, nonionic nature surface active agents, cholesterol varieties, saccharides such as mono and disaccharides, antioxidants are suggested for addition in the emulsion formulation.    iv. The process step of making the product sterile has not been specified.
In Japanese Patent 4-173736 (1992), a product containing 0.005% to 5% Amphotericin B, 0.5% to 25% phospholipid, preferably egg lecithin has been described. The composition has an average particle size diameter of 100 nm. This is not an emulsion and does not contain any oil phase.
In U.S. Pat. No. 5,389,373 (1995), a process of preparing an oil in water (o/w) emulsion of poorly soluble drugs is described. The process involves dissolving Amphotericin B in aqueous solution of high or low pH, adding the resulting solution of not more than 100 μg/ml strength to a preformed emulsion, adding to the emulsion an amount of acid, base or buffer appropriate to neutralise and to adjust the pH of the product to a desired value.
Drawbacks of this U.S. Pat. No. 5,389,373 (1995) process are:
The main weakness of this process is the limitation of the low strength of the Amphotericin B into the emulsion. In this process, Amphotericin B concentration is of the order of 100 μg/ml. Hence a larger volume of the composition is required to be injected which is therapeutically not advantageous.
In U.S. Pat. No. 5,534,502 (1996), Amphotericin B is decrystallized using an acid and ethanol and then homogeneously dispersed in a lipid, following which it is emulsified. In this process it is essential to dissolve Amphotericin B in ethanol, the most preferred quantity of ethanol is 400 to 600 ml/gm of Amphotericin B. The main weakness of this process is Amphotericin B is not stable in acid pH.
European Patent EP 0700678 (1996) describes a lipid emulsion which essentially contains citric acid or a pharmaceutically acceptable salt thereof and at least one member selected from the group consisting of methionine, phenylalanine, serine, histidine and pharmaceutically acceptable salts thereof, provided that it does not simultaneously contain methionine and phenylalanine.
It is an essential requirement to simultaneously use citric acid and at least one of the above amino acids. The process of preparation of an emulsion in accordance with this invention is described as follows:
Phospholipids and auxiliary agents for emulsification such as oleic acid are dissolved in an appropriate organic solvent such as hexane and then the solvent is distilled off under reduced pressure to give a lipid film. To the resulting lipid film, oil component and water and the mixture is preliminarily emulsified by vigorously stirring through shaking. The resulting liquid is emulsified using the currently used emulsifier. After completion of the emulsification the pH value of the resulting emulsion is adjusted to a predetermined level by addition of Hydrochloric acid or Sodium hydroxide. Then citric acid and amino acid are added to the emulsion to give a lipid emulsion. Alternatively the lipid emulsion likewise be prepared by adding an oil component and an aqueous solution of citric acid and amino acids to the lipid film prepared by the foregoing procedures and then subjecting the resulting mixture to the emulsification procedures.
Drawbacks of this European Patent EP 0700678 (1996) process are:
The process of making Amphotericin B emulsion has not been described specifically. Amphotericin B is one of the drugs from a list of about 70 drugs that is stated as “may be formulated in lipid emulsion”.    i) The process of preparing lipid emulsions involve dissolving phospholipids in appropriate organic solvents such as hexane.    ii) In the examples of this patent, amino acids and citric acid are added to the preformed lipid emulsions and the stability studied at 60° C. for discolouration. Intravenous emulsions are required to be stable to sterilisation temperatures of autoclaving.    iii) The process of sterilisation specified in one of the examples is heating at 60° C. for 1 hour and repetition of this sterilisation process three times every 24 hours. This process is not suitable for preparing intravenous injections.
The main object of the present invention is to develop a process for Amphotericin B emulsion useful for parenteral administration, having very low toxicity and to overcome the drawbacks and weaknesses of the prior art processes enumerated above.
Thus the principal part of the main object is to develop a process for coating solid powder of Amphotericin B with oil and placing the oil coated solid Amphotericin B powder in the oily phase of the oil-in-water emulsion with the provision for retaining the Amphotericin B incorporated in the oily phase of the emulsion throughout the entire process of manufacturing including autoclaving process of sterilisation and thereafter till its shelf life or use.
Another part of the main object is to develop a process for making such structured oil-in-water emulsion with average oil droplet in the emulsion controlled in the optimum range so that it is preferentially distributed in reticulo-endothelial system giving a low plasma concentration. Thus for injectable purpose it acts like aqueous outer phase emulsion carrying oil globules containing oil coated Amphotericin B.
Another part of the main object is to develop a process for making such structured Amphotericin B emulsion that will need only minimum of additives, which are essential for making an oil-in-water emulsion.